The neurotoxins produced by Clostridium botulinum (Botulinum Neurotoxins, BoNT) are among the most poisonous substances known. There are seven distinct serotypes of BoNTs (A-G), four of which are generally found to cause botulism in humans (A, B, E and F). There are several challenges in the diagnosis and treatment of botulism. Since the toxicity of BoNT is so great, it is necessary that BoNT can be detected at very low concentrations, preferably as the active toxin. The identification of the serotype is important for the most effective treatment. It is also imperative that the diagnosis be made rapidly since the equine-based treatment can have several substantial side effects.
The current standard for detecting botulinum toxins in, e.g., food, is the mouse lethality bioassay. Not only does this process require the use of animals, but also the process takes nearly a week to complete.
The current methods for detecting BoNT include a mouse bioassay and an enzyme-linked immunosorbent assay (ELISA). The mouse bioassay is currently the gold standard and is the only widely accepted method for the detection of BoNT. Mixtures of neutralizing antibodies are given to mice in conjunction with the sample in question to differentiate the toxin serotype. Mice receiving the appropriate anti-BoNT serotype antibody along with the toxic sample do not show symptoms and survive, while mice treated with the other serotype antibodies show symptoms and die. Importantly, this assay measures only active toxin. The mouse bioassay is very sensitive, detecting as little as 10 picograms (pg) of active toxin which is defined as 1 mouse LD50 or 1 unit of BoNT. However, the mouse bioassay can be slow (taking up to 4 days) for final results and it requires the sacrifice of many animals. It is highly desirable to have a more rapid technique of detecting botulinum toxins.
The ELISA is much more rapid, but is less sensitive, is problematic in certain matrices, shows cross reactivity between BoNT serotypes, and measures inactive toxin along with active toxin. The ELISA is currently used primarily as a fast screening technique and results are verified by the mouse bioassay.
Among recent approaches are: (1) U.S. Pat. No. 5,965,699 by Schmidt et al. wherein an assay for the proteolytic activity of type A botulinum toxin is described involving addition of a fluorigenic reagent that reacts with one of the proteolytic products to yield a fluorescent product that can be detected; and, (2) U.S. Pat. No. 6,762,280 by Schmidt et al. wherein assays for the proteolytic activity of clostridal neurotoxins are described involving synthetic peptide substrates modified with signal moieties such as fluorescent molecules for eventual signal output by a proteolytic product. Shine et al. describe still another fluorescence based detection method for botulinum neurotoxins in U.S. Pat. No. 6,504,006. Fluorescence arrays can however produce fluorescence response from unspecific cleavage of the substrate by other proteases than clostridal toxins.
Applicants have now developed a detection and differentiation method for botulinum neurotoxins based upon mass spectroscopy analysis of proteolytic products. The method can also allow for quantification of amounts of toxin and unequivocal product identification due to specific botulinum toxin cleavage of substrate.
It is an object of the present invention to provide such a detection and differentiation method for botulinum neurotoxins.